EP0144084A2 - Réactif pour dosage immunologique et procédé analytique utilisant ce réactif - Google Patents

Réactif pour dosage immunologique et procédé analytique utilisant ce réactif Download PDF

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Publication number
EP0144084A2
EP0144084A2 EP84114528A EP84114528A EP0144084A2 EP 0144084 A2 EP0144084 A2 EP 0144084A2 EP 84114528 A EP84114528 A EP 84114528A EP 84114528 A EP84114528 A EP 84114528A EP 0144084 A2 EP0144084 A2 EP 0144084A2
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Prior art keywords
liposomes
antibody
reagent
antigen
immunoassay
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EP84114528A
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German (de)
English (en)
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EP0144084A3 (fr
Inventor
Yoshio Ishimori
Masato Notsuki
Masao Koyama
Tatsuji Yasuda
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Toshiba Corp
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Toshiba Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/586—Liposomes, microcapsules or cells

Definitions

  • This invention relates to a reagent for immunoassay and an analytical method using the same, more particularly to a reagent for an immunoassay which is utilized to quantitatively analyze a specific antigen or antibody, and an analytical method in which the reagent is employed.
  • Typical examples of such tumor markers include a-fetoprotein (AFP), carcinoembryonic antigen (CEA), basic fetoprotein (BFP) and pancreatic oncofetal antigen (POA).
  • AFP a-fetoprotein
  • CEA carcinoembryonic antigen
  • BFP basic fetoprotein
  • POA pancreatic oncofetal antigen
  • radio immunoassay in which an antigen or antibody labelled by a radioactive material is employed.
  • the RIA method has been troublesome in points of handling and disposal treatment.
  • another immunoassay in which an antigen or antibody labelled with a variety of materials such as enzymes and fluorescent materials in place of the radioactive materials.
  • this immunoassay technique also has the drawback that a free antibody and a combined antibody must be separated from each other in any manner.
  • an EMIT method which has been disclosed in Rosenthal A. F., Vargas M. G. and Klass C. S. (1976), Clin. Chem., Vol. 22, p. 1899, is an epochal method by which measurement can be made in a uniform system without requiring any separation process, but it cannot be fundamentally applied to a protein antigen or antibody having a high molecular weight.
  • a protein is supported on the liposomes by using a bifunctional cross-linking agent such as glutaraldehyde.
  • a bifunctional cross-linking agent such as glutaraldehyde.
  • An object of this invention is to provide a method for an immunoassay of a homogeneous system by which an antigen or antibody in a sample can be quantitatively measured in a short time without any separating operations by using a developed reagent for the immunoassay in which a hydrophilic antigen or antibody is supported on liposomes.
  • the present inventors have intensively conducted researches to achieve the above-mentioned object, and as a result, they have now succeeded in immobilizing the antibody, at least a part of the antibody or antigen corresponding to the antigen or antibody in a sample on liposomes which will be dissolved by a function of complement, without lowering the activity of the antibody or antigen, and have found out that the object of this invention is achieved by enclosing a marker material in the liposomes. The completion of this invention has just been accomplished on the basis of these research results.
  • the reagent used with complement to analyse an antigen or antibody in a sample for immunoassay of this invention comprises liposomes; a hydrophilic antigen, antibody or at least a part of the antibody immobilized on the liposomes by a cross-linking method or an active lipid method; and a hydrophilic marker material enclosed in the liposomes.
  • this immunoassay comprises by mixing, with a sample including an antigen or antibody and a complement, a reagent for the immunoassay comprising liposomes, a hydrophilic antigen, antibody or at least a part of the antibody immobilized on the liposomes by a cross-linking method or an active lipid method, and a hydrophilic marker material enclosed in the liposomes; and measuring the marker material released from the liposomes in order to quantitatively determine the antigen or antibody concentration in the sample.
  • Liposomes for an immunoassay of this invention have a wide meaning including even a red blood cell ghost membrance.
  • any ones are acceptable so long as it has been conventionally used, but the liposomes composed of a phospholipid or glycolipid and cholesterol are more preferable from the viewpoint of the stability of the reagent.
  • the particularly stable liposomes can be prepared on the condition that the molar ratio of phospholipid : cholesterol is between 1 : 0.1 and 1 : 5.0, more preferably 1 : 1 or so.
  • the number of its carbon atoms is preferably 12 to 18, and it is more preferable that this number is even.
  • the molar ratio of these lipids to cholesterol is preferable in the range as mentioned above.
  • Antigens, antibodies or at least a part of the antibody to be immobilized on the liposomes include antigens for tumor markers (the above-mentioned AFP, BEP, CEA, POA, etc.), immunoglobulins (IgA, IgE, IgG, IgM, etc.), hormones (insulin, T 3 , T41 etc.), drugs (phenitoin, digoxin, etc.) and the like; or antibodies against the aforesaid antigens.
  • tumor markers the above-mentioned AFP, BEP, CEA, POA, etc.
  • immunoglobulins IgA, IgE, IgG, IgM, etc.
  • hormones insulin, T 3 , T41 etc.
  • drugs phenitoin, digoxin, etc.
  • these antigens, antibodies or at least a part of the antibodies are required to be hydrophilic.
  • the antigen, antibody or at least a part of the antibody is immobilized on the liposomes by using a cross-linking agent or an activator for a lipid with the aid of a covalent bond between atoms.
  • an amount of the phospholipid and/or glycolipid constituting the liposomes is preferably such that an amount of the above compound which has reacted with the cross-linking agent is within the range of 0.01 to 30 mol %, in the case a cross-linking method is employed. Further, it is preferred that a concentration of the hydrophilic antigen, antibody or at least a part of the antibody to be immobilized is within the range of 0.01 to 20 mg/ml with respect to 0.5 mM of the liposomes in terms of the lipid.
  • the antigen, antibody or at least a part of the antibody is immobilized on the liposomes by using an undermentioned cross-linking agent such as N-succinimidyl-3-(2-pyridyldithio)propionate with the aid of a covalent bond between atoms.
  • an undermentioned cross-linking agent such as N-succinimidyl-3-(2-pyridyldithio)propionate with the aid of a covalent bond between atoms.
  • the marker material to be enclosed in the liposomes must be hydrophilic and must be determinable when released out of the liposomes.
  • marker materials include fluorescent compounds such as carboxy fluorescein which emits no fluorescence due to self quenching at a high concentration but gives off a very intensive fluorescence at a low concentration (10- 3 M or less); luminous compounds-such as luminol and luciferin which emit light owing to an oxidative reaction outside the liposomes; saccharides such as glucose and sucrose which are decomposed by the function of an oxidative fermentation in order to lead to the consumption of oxygen or the production of hydrogen peroxide; relatively great ionic compounds such as tetrapentyl ammonium; coenzymes such as nicotinamidoadeninedinucleotide (NAD); radical compounds such as methylviologen; absorptive compounds having absorption bands specific to a visible region or ultraviolet region such as water-soluble metallic indicators, e.g.
  • Xylenol Orange (XO), Methylxylenol Blue (MXB), Methylthymol Blue (MTB), water-soluble PADAP (5-Br-PAPS), water-soluble PADAB (5-Br-PSAA), Bismuthiol II, Pyrocate- chol Violet (PV) and Eriochrome Black T (BT), and water-soluble dyes, e.g. Rhodamine B, Oxamine Red X and Acid Orange I; and enzymes such as alkaline phosphatase, glucose oxidase, peroxidase, luciferase and the like.
  • the reagent for the immunoassay regarding this invention can react with the antigen or antibody to be detected, which is included in the sample, and the separately added complement in order to discharge a marker material from inside of the liposomes. Then, the discharged maker material is measured by means of a method with respect to the discharged marker material, whereby the quantitative analysis of the antibody or antigen included in the sample can easily be carried out.
  • the reagent for the immunoassay of this invention described hereinbefore can be prepared, for example, in the following method: First of all, a desired lipid is reacted with a cross-linking agent (a method of using such a cross-linking agent is called a cross-linking method) in a solvent (in place of the cross-linking agent, an activator for the lipid may be used, and a method of using such an activator is called an active lipid method), and functional groups which will be able to combine with the antigen, antibody or at least a part of the antibody immobilized on the liposomes are introduced into the lipid molecules.
  • a cross-linking agent a method of using such a cross-linking agent
  • an activator for the lipid in a solvent
  • an active lipid method an activator for the lipid
  • the thus obtained functional lipid, cholesterol and, if necessary, another lipid are placed in a flask, and a solvent is further added thereto. After mixing, evaporation for the removal of the solvent and suction to dryness are followed. Subsequently, a selected aqueous marker material solution is added to the flask on the inner wall surface of which a thin membrane is formed, and it is sealed with a cap. After stirring, a suspension of the immobilized liposomes is prepared.
  • the antigen, antibody or at least a part of the antibody to be immobilized on the liposomes is reacted with the cross-linking agent in a buffer solution to introduce cross-linking groups thereinto.
  • the antigen or antibody is further reacted with a reagent (e.g., dithiothreitol (DTT)) for reducing the cross-linking groups, thereby obtaining the modified antigen, antibody or at least a part of the antibody.
  • DTT dithiothreitol
  • the immobilized liposomes are reacted with the modified antigen, antibody or at least a part of the antibody (in the case that the active lipid method is employed, the unmodified antigen, antibody or at least a part of the antibody) in the buffer solution in order to prepare the reagent for the immunoassay of this invention.
  • a reagent is obtained in the form of liposomes which include the marker material therein and support the immobilized antigen, antibody or at least a part of the antibody on the surfaces thereof.
  • cross-linking agents used in the above-mentioned cross-linking method include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB), N-succinimidyl-4-(p-maleimidophenyl)acetate (SMPA), N-succinimidyl-4-(p-maleimidophenyl)propionate (SMPP), N-(y-maleimidobutyryloxy)-succinimide (GMB6) and N-(E-maleimidocaproyloxy)succinimide (EMCS).
  • SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
  • SMPB N-succinimidyl-4-(p-maleimidophenyl)butyrate
  • SMPA N-succin
  • the above compound SPDP is represented by the following formula:
  • This compound SPDP reacts under moderate conditions to couple compounds having primary amino groups to each other, and it is commercially available from Pharmacia Inc.
  • This cross-linking agent can be utilized, for example, as follows: The protein antigen to be immobilized is treated with the agent SPDP and is reduced with dithiothreitol (DTT). Then, it is reacted with the liposomes which has previously been treated with SPDP. After allowed to stand at room temperature or less for a period of several hours to one day, the antigen can be immobilized on the liposomes.
  • DTT dithiothreitol
  • the above-mentioned compound SMPB is represented by the following formula:
  • This compound SMPB can immobilize a protein by a reaction similar to that of SPDP.
  • an -S-S- bond is not included in the end product (an -S-bond is only included therein).
  • the end product is stable even under a reducing atmosphere such as in a serum.
  • examples of the activators for the lipids include cyanogen bromide (CNBr), cyanuric chloride (CC), epichlorohydrin (EH), O-bromoacetyl-N-hydroxysuccinimide and l,4-bis(2,3-epoxypropoxy)butane (BEPB).
  • CNBr, CC, EH and BEPB serve to activate compounds having saccharide residues and to couple them to compounds having primary amino groups. Therefore, when the saccharide residues are present on the liposomes, these reagents can be applied.
  • the antigen itself is a glycoprotein and the primary amino groups are present on the liposomes, these activators are likewise effective.
  • the immobilization of the hydrophilic antigen, antibody or at least a part of the antibody on the liposomes will be feasible, though such an immobilization has been impossible in former days.
  • a strong cross-linking agent e.g., glutaraldehyde (hereinafter referred to as GA).
  • the reagent for the immunoassay of this invention may be manufactured by first coupling the lipid to the antigen, antibody or at least a part of the antibody with the aid of the cross-linking agent or the activator for the lipid; adding the coupled material together with a surface active agent to water in order to form micells; and removing the surface active agent therefrom by means of dialysis or gel filtration.
  • Materials which can be determined by the a-bove-mentioned reagent for the immunoassay widely range and include antigens of tumor markers (the above-mentioned AFP, BEP, CEA, POA, etc.), immunoglobulins (IgA, IgE, IgG, IgM, etc.), hormones (insulin, T , T 4 , etc.), drugs (phenitoin, digoxin, etc.) and the like; and antibodies or at least a part of the antibodies corresponding to the aforesaid antigens.
  • tumor markers the above-mentioned AFP, BEP, CEA, POA, etc.
  • immunoglobulins IgA, IgE, IgG, IgM, etc.
  • hormones insulin, T , T 4 , etc.
  • drugs phenitoin, digoxin, etc.
  • materials to be detected which will bring about an antigen-antibody reaction with the immobilized antigen, antibody or at
  • the reagent for the immunoassay described above is mixed with a sample including the antigen or antibody (if the antigen is immobilized on the liposomes, the antibody sample will be used; if the antibody or at least a part of the antibody is done, the antigen sample will be used) and a complement in a suitable buffer solution (e.g., a gelatin-Veronal buffer solution [hereinafter referred to as GVB - ] including, at ultimate concentrations, 0.1 mM MgC1 2 and 0.15 mM CaCl 2 , respectively) in order to bring about a coupling reaction of the antigen-antibody with the complement.
  • a suitable buffer solution e.g., a gelatin-Veronal buffer solution [hereinafter referred to as GVB - ] including, at ultimate concentrations, 0.1 mM MgC1 2 and 0.15 mM CaCl 2 , respectively
  • a marker material is released out of the liposomes in proportion of their reacting amount:
  • determination is carried out by an analytical method (e.g., if the marker material has fluorecsence, a fluorescent analytical method will be emploed) in accordance with this marker material, and an amount of the antigen or antibody in the sample can be measured on the basis of a previously prepared calibration curve.
  • the complements used in the determining operation are not particularly limited, but the preferred complements each have a high activity, i.e. a high complement value and preferably in the range from 0.1 to 10 HC 50 .
  • a guinea pig serum is preferably employed.
  • sera of rabbit, mouse, human and the like may also be applicable.
  • the complement value is an important factor to decide a measurement range and a detection limit, and better measurement results can be obtained by changing this complement value variously.
  • an enzyme or substrate in a sample can also be quantitatively analyzed.
  • the hydrophilic antigen, antibody or at least a part of the antibody is immobilized on the liposomes without deteriorating its activity, and since the marker material is enclosed in the liposomes, the accurate determination, having a high detection sensitivity, of the antigen or antibody can be carried out in a homogeneous system in a short time.
  • the analytical method of this invention can be applied to an extensive region of the materials to be detected and can be accomplished at lower costs. Further, the present invention permits facilitating a highly accurate automatic analysis in miniature and simultaneously measuring many components.
  • DPPC dipalmitoylphosphatidyl choline
  • DPPE dipalmitoylphosphatidyl ethanolamine
  • DTT dithiothreitol
  • SPDP N-succinimidyl-3-(2-pyridyldithio)-propionate
  • SPDP Sephadex G-25 Fine
  • Other reagents were commercially available ones (special grade), which were directly used without any purification. Further, ionexchange water was used as water.
  • a small amount of a buffer solution (GVB including, at ultimate concentrations, 0.1 mM MgCl 2 and 0.15 mM CaCl 2 , respectively; hereinafter merely referred to as GVB ) was added thereto, and all of the liposomes suspension was transferred to a centrifuge tube. Centrifugal treatment was carried out at 4 °C and 15,000 rpm for 20 minutes in order to remove free carboxyfluorescein. This treatment was repeated using GVB 2+ till the supernatant became transparent. In the last place, 2 ml of GVB 2+ and 5 ul of 10 % NaN 3 were added thereto and were suspended by means of the Vortex mixer. After introducing nitrogen and sealing, the product was stored in a refrigerator.
  • VVB buffer solution
  • a first peak fraction (about 2 ml) were further added 2 ml of the acetic acid buffer solution, and after replaced with nitrogen, about 30 mg of dithiothretol were added thereto. Reaction was carried out over enough stirring for 20 minutes. After the reaction, the reaction mixture was developed through a column (gel volume : about 30 ml) filled with Sephadex G-25 Fine gel which was previously saturated with a 0.01 M HEPES buffer solution, and elution was carried out with the HEPES buffer solution. A first peak fraction (about 2 ml) was collected, and after nitrogen replacement, the product was stored in a refrigerator till used.
  • the liposomes suspension prepared above was mixed with an equal amount of the modified human IgG solution, and after nitrogen replacement and sealing, reaction was carried out overnight at room temperature with slowly shaking, followed by washing with the HEPES buffer solution and next with GVB 2+ in order to remove unreacted human IgG.
  • GVB 2+ in an amount equal to that of the liposomes suspesion used in the reaction, and 5 ul of 10 % N aN 3 were lastly added, and after suspending and nitrogen replacement, the product was stored in a refrigerator till used.
  • the liposomes including carboxyfluorescein was prepared. After centrifugal washing, the liposomes were suspended in a 0.1 M phosphoric acid buffer solution (including 0.85 % of NaCl; pH 6.5) (2 ml), and 150 mg of DTT were then added thereto. After nitorgen replacement, stirring was sufficiently carried out and reaction was done at room temperature for 2 hours in situ. Washing was carried out (by using GNV 2+ already replaced with nitrogen) centrifugally (15,000 rpm, 20 minutes) and 5 ul of 10 % NaN 3 were added thereto, and the resulting product was stored in a refrigerator.
  • a phosphoric acid buffer solution including 0.85 % of NaCl; pH 6.5
  • the liposomes which will be applied to the coupling reaction with modified human IgG is preferably treated with DTT just before the coupling step, but in the case that the liposomes coolingly stored is unavoidably used, it is preferred that a small amount (10 to 20 mg) of DTT is added thereto again before the coupling reaction and the reaction is then carried out at room temperature for 30 minutes or so. This process serves to cut off S-S bonds formed between the liposomes during the storage.
  • Example 1 Following the same procedure as in Example 1, an anti-human IgG antibody was measured using the liposomes, and results similar to those of Example 1 were obtained.
  • the liposomes, prepared in Example 1, on which human IgG was immobilized was utilized to measure an amount of human IgG in the human serum.
  • a 1,000-fold, a 10,000-fold and a 20,000-fold diluted (by using GVB 2+ ) anti-human Ig G antibody were poured every 25 ul, and samples of human sera which were diluted 10 to 10 6 times were added thereto every 25 ul. Reaction was then carried out overnight at 4 °C.
  • Example 3 Human AFP (commercially available from Nippon Biotest Laboratory) was immobilized on the surface of liposomes including carboxyfluorescein.
  • a suitably diluted (100 to 1000 ng/ml) (by using GVB 2+ ) anti-human AFP antibody solution was poured every 25 U l into wells on a microtiter plate, and a human AFP standard solution (3 to 1000 ng/ml; available from Nippon Biotest Laboratory) were added every 5 ul to each pair of two wells. Then, reaction was carried out at room temperature (about 25 °C) for 30 minutes.
  • the liposomes were prepared in the same manner as in Example 1, and 5 mg of human IgG were added to this liposomes suspension. Reaction was carried out at room temperature for 3 hours. After washing with GVB - , the liposomes were suspended in 2 ml of GVB again and were then preserved in a refrigerator after the introduction of nitrogen.
  • Example 1 The procedure of the liposomes preparation in Example 1 was repeated with the exception that an amount of DPPE-DTP was 50 ul and an equal amount of 0.2 M water-soluble PADAP (5-Br-PAPS) was substituted for carboxy fluorescein.
  • Example 1 The procedure of the Example 1 was repeated with the exception that 5 mg of human IgG (available from Miles-Yeda Inc.) were used, and elution was carried out with the HEPES buffer solution. An initial protein fraction (about 2 ml) was collected, and after nitrogen replacement, it was stored in a refrigerator till used.
  • human IgG available from Miles-Yeda Inc.
  • Example 1 The same procedure in Example 1 was repeated in order to prepare the liposomes on which human IgG was immobilized. (3) Measurement of an anti-human IgG antibody by the use of the human IgG immobilized liposomes
  • GVB 2+ (GVB - including, at ultimate concentrations, 0.1 mM MgCl 2 and 0.15 mM CaCl 2 , respectively) having pH 7.8 was poured into cells every 25 ul.
  • the above-mentioned liposomes suspension was diluted with GVB 2+ 100-fold and was poured into each cell every 5 ⁇ l.
  • a complement (guinea pig serum) suitably diluted with GVB 2+ was added thereto every 25 ⁇ l, and reaction was carried out at 37 °C for an hour. Measurement of the optical density was made at an absorption wavelength of 550 nm.
  • the measured values were represented with relative values by regarding, as 100 %, a difference between an absorption in the case that Triton X-100 and GVB 2+ were used in an amount of 25 ul, respectively, in place of the antibody and complement and an absorption in the case that 25 ⁇ l of GVB 2+ were used in place of the antibody.
  • This Figure 3 indicates that the anti-human IgG antibody can be determined within the range of 10 -3 to 1 0 - 5 mg/ml.
  • the curve a in Figure 4 shows-a relation between a concentration of the anti-human IgG antibody included in the sample and a relative release rate of the marker material which was-released by the reaction of the reagent for the immunoassay of this invention.
  • a definite relative relation as present between both of these factors, and thus the quantitative analysis of the sample is feasible on the basis of this fact.
  • the curve b in the drawing exhibits a relation therebetween in the case that glutaraldehyde (GA) disclosed in conventional techniques was used as the cross-linking agent and the liposomes comprising DPPC, cholesterol and DPPE-DTP in a molar ratio of 1 : 1 : 0.05 were employed.
  • G glutaraldehyde
  • the liposomes in which the conventional cross-linking agent was used scarcely change the release proportion of the marker material in accordance with the variation of the sample concentration. By this fact, it has been confirmed that such liposomes cannot be applied to an accurate quantitative analysis.
  • human IgG immobilized liposomes were prepared by changing a content of DPPE-DTP in the liposomes, in other words, by varying an amount of the lipid reacted with a cross-linking agent.
  • the results are shown in Figure 5. As be apparent from this drawing, the release of CF was not observed at all in the case of the liposomes including no DPPE- DTP .
  • a usable range was from 0.01 mol, and when the content of DPPE-DTP was increased, the CF release rate increased up to 1 mol %. However, when it was added in excess of this level, the CF release rate did not vary. Further, in the case of the liposomes including 30 mol % or more of DPPE-DTP, the spontaneous CF release was noticeably observed, and the stability was poor. By the results of this example, it was confirmed that constitutional ratios of the phospholipid and glycolipid reacted with the cross-linking agent are within the range of 0.01 to 30 mol %.
  • Example 7 a concentration of human IgG was variously changed when it was immobilized on the liposomes comprising DPPC, cholesterol and DPPE-DTP in a molar ratio of 1 : 1 : 0.05, with the intention of inspecting a reactivity of the anti-human IgG antibody with the liposomes.
  • a reactivity between the human IgG immobilized liposomes prepared in Example 7 and 2 x 10 - 3 mg/ml of the anti-human IgG antibody was inspected by employing various complement values. As shown in Figure 7, when the complement values were 0.1 CH 50 , CF was released from almost all the liposomes even under conditions including no antibody. Therefore, it was impossible to detect the antibody.
  • a measurement system can be constituted in compliance with a material to be detected and its concentration.
  • antigens could be determined in a similar manner.
  • FIG. 10 shows the relation between a relative release rate and an antibody dilution rate, which relative release rate was obtained by regarding, as 100 %, a value obtained in the case that 10 % Triton X-100 was used.

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EP84114528A 1983-11-30 1984-11-30 Réactif pour dosage immunologique et procédé analytique utilisant ce réactif Withdrawn EP0144084A3 (fr)

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JP58224509A JPH06100601B2 (ja) 1983-11-30 1983-11-30 免疫分析用試薬及びそれを用いた分析方法
JP224509/83 1983-11-30

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EP0144084A2 true EP0144084A2 (fr) 1985-06-12
EP0144084A3 EP0144084A3 (fr) 1988-07-27

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987002777A1 (fr) * 1985-10-24 1987-05-07 Research Corporation Limited Reactif biochimique
FR2595141A1 (fr) * 1986-02-28 1987-09-04 Inst Nat Sante Rech Med Nouvelles compositions de liposomes contenant un des elements d'une reaction affine, procede pour leurs preparations et utilisation de ces compositions pour la detection de l'element complementaire de la reaction affine, notamment d'un antigene parasitaire
EP0248621A2 (fr) * 1986-05-31 1987-12-09 Kabushiki Kaisha Toshiba Réactif pour l'immuno-essai
GB2200448A (en) * 1987-01-23 1988-08-03 Univ London Targetted liposomes and their use in immunoassay
EP0307175A2 (fr) * 1987-09-09 1989-03-15 Ciba Corning Diagnostics Corp. Méthode pour la préparation de conjugués de diesters phosphoniques utiles dans la préparation de liposomes immuno-actifs
EP0312212A1 (fr) * 1987-09-21 1989-04-19 Kabushiki Kaisha Toshiba Immobilisation d'une substance bioactive sur une composition de lipide contenant un composé modifié de lipide
EP0313666A1 (fr) * 1987-05-06 1989-05-03 Teijin Limited Procede et kit d'immunoanalyse utilisant des liposomes
WO1990007924A1 (fr) * 1989-01-19 1990-07-26 Micro Vesicular Systems, Inc. Couplage de proteines a des vesicules de lipides
US5053497A (en) * 1984-12-28 1991-10-01 Technion Instruments Corporation Phospholipid conjugates and their preparation
US5081048A (en) * 1988-03-11 1992-01-14 The Nisshin Oil Mills, Ltd. Agent for the determination of antigen or antibody, and a device and measuring method related thereto
WO1992007876A1 (fr) * 1990-10-24 1992-05-14 Henning Berlin Gmbh Chemie- Und Pharmawerk Conjugue ligand-proteine de transport
US5449556A (en) * 1988-08-01 1995-09-12 Ciba Corning Diagnostics Corp. Method for detection of an analyte using acridinium esters and liposomes
US5538901A (en) * 1988-09-26 1996-07-23 Ciba Corning Diagnostics Corp. Nucleophilic polysubstituted aryl acridinium ester conjugates uses thereof
US5663074A (en) * 1988-09-26 1997-09-02 Chiron Diagnostics Corporation Nucleophilic polysubstituted aryl acridinium ester conjugates and syntheses thereof
US6132763A (en) * 1988-10-20 2000-10-17 Polymasc Pharmaceuticals Plc Liposomes

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JPS6250665A (ja) * 1985-08-30 1987-03-05 Denka Seiken Co Ltd 肺炎マイコプラズマ抗体価の測定法
EP0247497B1 (fr) * 1986-05-20 1992-03-04 Wako Pure Chemical Industries, Ltd. Liposomes portant des groupes fonctionnels et leur procédé de production
JP2668403B2 (ja) * 1988-08-02 1997-10-27 日水製薬株式会社 Ａｔｌ抗体測定試薬
EP0382400B1 (fr) * 1989-02-06 1996-03-20 Wako Pure Chemical Industries Ltd Procédé pour l'essai immunologique et réactifs liquides pour cet usage

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Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5053497A (en) * 1984-12-28 1991-10-01 Technion Instruments Corporation Phospholipid conjugates and their preparation
US5122450A (en) * 1985-10-24 1992-06-16 Research Corporation Limited Biochemical reagent
WO1987002777A1 (fr) * 1985-10-24 1987-05-07 Research Corporation Limited Reactif biochimique
FR2595141A1 (fr) * 1986-02-28 1987-09-04 Inst Nat Sante Rech Med Nouvelles compositions de liposomes contenant un des elements d'une reaction affine, procede pour leurs preparations et utilisation de ces compositions pour la detection de l'element complementaire de la reaction affine, notamment d'un antigene parasitaire
EP0248621A2 (fr) * 1986-05-31 1987-12-09 Kabushiki Kaisha Toshiba Réactif pour l'immuno-essai
EP0248621A3 (en) * 1986-05-31 1989-06-07 Kabushiki Kaisha Toshiba Reagent for immunoassay
GB2200448B (en) * 1987-01-23 1991-01-16 Univ London Targetted liposomes and their use in immunoassay
GB2200448A (en) * 1987-01-23 1988-08-03 Univ London Targetted liposomes and their use in immunoassay
US5000960A (en) * 1987-03-13 1991-03-19 Micro-Pak, Inc. Protein coupling to lipid vesicles
EP0313666A1 (fr) * 1987-05-06 1989-05-03 Teijin Limited Procede et kit d'immunoanalyse utilisant des liposomes
EP0313666A4 (fr) * 1987-05-06 1990-03-12 Teijin Ltd Procede et kit d'immunoanalyse utilisant des liposomes.
US5173406A (en) * 1987-05-06 1992-12-22 Teijin Limited Liposome immunoassay method and kit therefor
EP0307175A2 (fr) * 1987-09-09 1989-03-15 Ciba Corning Diagnostics Corp. Méthode pour la préparation de conjugués de diesters phosphoniques utiles dans la préparation de liposomes immuno-actifs
EP0307175B1 (fr) * 1987-09-09 1995-03-22 Ciba Corning Diagnostics Corp. Méthode pour la préparation de conjugués de diesters phosphoniques utiles dans la préparation de liposomes immuno-actifs
EP0312212A1 (fr) * 1987-09-21 1989-04-19 Kabushiki Kaisha Toshiba Immobilisation d'une substance bioactive sur une composition de lipide contenant un composé modifié de lipide
US5080833A (en) * 1987-09-21 1992-01-14 Kabushiki Kaisha Toshiba Immobilization of bioactive substance on lipid composition containing modified lipid compound
US5081048A (en) * 1988-03-11 1992-01-14 The Nisshin Oil Mills, Ltd. Agent for the determination of antigen or antibody, and a device and measuring method related thereto
US5449556A (en) * 1988-08-01 1995-09-12 Ciba Corning Diagnostics Corp. Method for detection of an analyte using acridinium esters and liposomes
US5595875A (en) * 1988-08-01 1997-01-21 Ciba Corning Diagnostics Corp. Method for detection of an analyte using acridinium ester and liposomes
US5656500A (en) * 1988-08-01 1997-08-12 Chiron Diagnostics Corporation Luminescent conjugates for use in luminescent assays
US5538901A (en) * 1988-09-26 1996-07-23 Ciba Corning Diagnostics Corp. Nucleophilic polysubstituted aryl acridinium ester conjugates uses thereof
US5663074A (en) * 1988-09-26 1997-09-02 Chiron Diagnostics Corporation Nucleophilic polysubstituted aryl acridinium ester conjugates and syntheses thereof
US6080591A (en) * 1988-09-26 2000-06-27 Bayer Corporation Nucleophilic polysubstituted aryl acridinium ester conjugates and syntheses thereof
US6132763A (en) * 1988-10-20 2000-10-17 Polymasc Pharmaceuticals Plc Liposomes
WO1990007924A1 (fr) * 1989-01-19 1990-07-26 Micro Vesicular Systems, Inc. Couplage de proteines a des vesicules de lipides
WO1992007876A1 (fr) * 1990-10-24 1992-05-14 Henning Berlin Gmbh Chemie- Und Pharmawerk Conjugue ligand-proteine de transport

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EP0144084A3 (fr) 1988-07-27
JPH06100601B2 (ja) 1994-12-12

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